This present invention relates generally to sample holders for measuring instruments and specifically to sample holders for instruments which utilize surface plasmon resonance (SPR) for measuring chemical or biochemical compositions. Surface plasmon resonance is being used in biosensing, in such areas as immunoassay and nucleic acid detection Basically, surface plasmons are electromagnetic waves created along an interface between a conducting material and a non-conducting material. A common technique for their creation is to direct a beam of electromagnetic radiation into a glass prism with an angle of incidence above the critical angle so that it undergoes total internal reflection. The internal reflection creates an evanescent electromagnetic wave at a region outside of the prism adjacent to the surface. When a thin conductive film is deposited on the surface of the prism, surface plasmons will be formed.
Surface plasmon resonance occurs when the momentum (or the wave vector) and energy (i.e. frequency) of the evanescent electromagnetic wave are made to match the momentum and energy of the surface plasmons respectively. It is characterized by a sharp decrease in intensity of the reflected beam as its energy is transferred, because of the resonance, to the surface plasmons.
The wave vector Ke of the evanescent wave is defined by the equation:Ke=(ω/C)n sin Θ,
where ω is the angular frequency of the incident beam, c is the speed of light in vacuum, n is the refractive index of glass and Θ is the angle of incidence. The wave vector of the surface plasmon is defined by the equation:Ksp=(ω/c)(1/εm+1/εs)−½,
where εm is the real part of the dielectric constant of the metal and εs is the dielectric constant of the substance under test (or in the absence of any substance, of air) surrounding the metal.
At resonance, the wave vector of the evanescent wave is the same as that of the surface plasmons so that there is no electromagnetic wave reflected from the surface. Therefore, occurrence of the surface plasmon resonance is given by the equation:Ke=Ksp.
If a periodic structure such as a grating or a surface acoustic wave is impressed upon the thin metal layer, the above equation becomes:Ke+k=Ksp.
where k is the wave vector due to the periodic structure.
The above equation provides a useful tool for measuring differences between the values of εs of different materials. It also provides a useful tool for detecting the presence of trace surface chemicals in a substance that alters its εs value. By measuring the differences of Ke at resonance, the changes in εs can be determined.
Surface plasmon resonance measuring instruments typically utilize the above equality condition and measure the differences of Ke by varying Θ and sensing the reflected beam at different values of Θ to detect the resonance. In these surface plasmon resonance measuring instruments, sensing the reflected beam at different values of Θ has been accomplished by various methods. Other surface plasmon resonance measuring instruments utilize the above equality condition and measure the differences of Ke by varying parameters and sensing the reflected beam at different values of the varied parameter.
In the typical surface plasmon resonance measuring instrument, the sample to be measured are placed in a pattern of droplets on a gold surface of a glass slide. The gold coated slide is referred to as a chip.
The present invention is directed generally to surface Plasmon Resonance technology (SPR) which allows the characterization of bio specific interactions of label-free compounds. The invention is specifically directed to a variation of SPR known as the Kretchmann process and, more specifically, to the substrate or chip that is part of the process. In the Kretchmann process, a collimated beam of light, e.g. laser, is directed through a prism to a chip that is supported on the prism. The chip is glass slide coated with metal such as silver or gold. The light hits the glass gold interface. At a specific angle, the light will be absorbed. The nonabsorbed light is reflected back through the prism and detected. Biological samples to be analyzed are deposited on the gold in an array of droplets. Each droplet has a target biological element bound to a ligand, for example, that will have an effect on the reflected light to the detector which will be indicative of the specific biological element in the droplet.
More specifically, the Kretchman process uses a constant wave-length light source, e.g. laser. The laser light is directed through a P-polarizer to pass only the P-polarized light. The light is then directed through a prism. A glass slide coated with gold is positioned fixed on the hypotenuse of the prism with refractive index matching fluid. The light hits the glass gold interface. At a specific angle, the light will be absorbed. The non-absorbed light is reflected and detected using a photomultiplier tube or CCD camera.
One of the problems associated with conventional chips is that as the droplet spreads over the gold surface and as the droplet dries, the analyte migrates to the periphery of the droplet, so that there is a higher concentration of biological material at the periphery of the droplet than in the body of the droplet. This non-uniform distribution of analyte in the droplet, or “peripheral concentration effect”, can produce a distortion of the light signal received by the detector, and interferes with the ability of the device to correctly analyze the content of the droplet.
What is needed is a sample holder for an instrument that can be used for measuring chemical compositions by measuring the dielectric constants thereof utilizing surface plasmon resonance, said sample holder being designed so that the measurement distorting result of the sample droplet “peripheral concentration effect” is minimized or eliminated.